Technical innovation has transformed our ability to analyze genetic information in a comprehensive fashion. DNA biochips and related technologies now permit the simultaneous measurement of the structures and activities of essentially all human genes. This comprehensive capability has given us our first glimpse of the complexity of the underlying molecular events that define metabolism and disease pathogenesis. To complement this information and translate its findings to the diagnosis and treatment of human disease, these observations must be translated and extended by direct measurement of the proteins that are encoded by this genetic information. Within the pharmaceutical, biotechnology, and research communities, there is currently a large unmet need for multiplexed protein detection and quantification technologies. The current techniques for multiplexed protein profiling rely heavily on application of sandwich-ELISA format in miniaturized scale. However, these techniques suffer limited multiplexing capabilities and variable performance including specificity, sensitivity, and accuracy. The innovation described in this proposal is designed to alleviate these limitations by eliminating the need for the use of sandwich-ELISA in microarray assays and introducing signal amplification mechanism to improve the assay performance. This innovation, termed the protein footprint scanning technology, is founded on a novel immunochemical detection method which combines the specificity of antibodies with regiospecific amino acid cross-linking chemistry to produce analyte-specific quantification. The successful validation and implementation of this technology will catalyze the development of highly sensitive protein microarray assays using only one antibody per target analyte. This will in turn enable microarrays with higher content multiplexity, diversity, and novelty. The expanded content will be especially important for cancer research since the multi-factorial nature of oncogenesis will likely require parallel examination of large numbers of proteins in cellular and extracellular proteome compartments. As a proof-of-concept, the feasibility of applying this innovative technology for protein detection and quantification will be validated using multiplexed protein microarray assays consisting of 10 validated cancer biomarkers.